Green synthesis, characterization, and biological evaluation of gold and silver nanoparticles using Mentha spicata essential oil

Green synthesis of bioactive nanoparticles (NPs) is getting more attractive in various fields of science including the food industry. This study investigates the green synthesizing and characterization of gold NPs (AuNPs) and silver NPs (AgNPs) produced using Mentha spicata L. (M. spicata) essential oil as well as their antibacterial, antioxidant, and in vitro cytotoxic effects. The essential oil was mixed with both Chloroauric acid (HAuCl4) and aqueous silver nitrate (AgNO3) solutions separately and incubated at room temperature for 24 h. The chemical composition of the essential oil was identified by gas chromatography coupled with a mass spectrometer detector (GC–MS). Au and Ag nanoparticles were characterized using UV–Vis spectroscopy, transmission electron microscopy, scanning electron microscopy, dynamic light scattering (DLS), X-ray diffraction (XRD) and Fourier transform infrared (FTIR). The cytotoxicity of both types of nanoparticles was evaluated using MTT assay on cancerous HEPG-2cell line by exposing them to various concentrations of both NPs for 24 h. The antimicrobial effect was evaluated by the well-diffusion technique. The antioxidant effect was determined by DPPH and ABTS tests. According to the results of GC–MS analysis, 18 components were identified, including carvone (78.76%) and limonene (11.50%). UV–visible spectroscopy showed a strong absorption peak of 563 nm and 485 nm, indicating the formation of Au NPs and Ag NPs, respectively. TEM and DLS demonstrated that AuNPs and AgNPs were predominantly spherical shaped with average sizes of 19.61 nm and 24 nm, respectively. FTIR analysis showed that biologically active compounds such as monoterpenes could assist in the formation and stabilization of both types of NPs. Additionally, XRD provided more accurate results, revealing a nano-metal structure. Silver nanoparticles exhibited better antimicrobial activity against the bacteria than AuNPs. Zones of inhibition ranging 9.0–16.0 mm were recorded for the AgNPs, while zones of 8.0–10.33 mm were observed AuNPs. In the ABTS assay, the AuNPs and AgNPs showed a dose-dependent activity and synthesized nanoparticles exhibited higher antioxidant activity than MSEO in both assays. Mentha spicata essential oil can be successfully used for the green production of Au NPs and Ag NPs. Both green synthesized NPs show antibacterial, antioxidant, and in vitro cytotoxic activity.


Results and discussion
Gas chromatography-mass spectrometry and UV-Vis spectroscopy. In this work, Au NPs and Ag NPs were successfully synthesized by using Mentha spicata essential oil as demonstrated by the color change of the reaction medium to ruby-red and light brown, respectively (Fig. 1a,b) which is cause of the effect of surface plasmon resonance and is in agreement with the results of Thanighaiarassu 18 , Muniyappan and Nagarajan 19 , Wang, Xu 20 and Erci, Cakir-Koc 21 . Previous studies reported that the secondary metabolites such as flavonoids, phenolic acids, tannins, vitamins, proteins, etc. act as reducing and capping agents and are responsible for synthesis of MNPs. The presence of strong reducing phytochemicals in the plant extract not only promotes a fast reduction rate, but also determines the size distribution and morphology of metal nanostructures 22 .
The absorbance of both NPs at different wavelengths was investigated by UV-Vis spectroscopy for 24 h after the addition of essential oil to HAuCl 4 and AgNO 3 solutions. The maximum absorption at 563 nm was seen after 24 h, representing the absorption peak of Au NPs while the highest absorption band was revealed at 485 nm for Ag NPs (Fig. 2a,b). These results were in parallel with the results of Khatami, Soltani Nejad 23 and Erci, Cakir-Koc 21 . The study of Soltani Nejad 24 showed the absorption peak of the UV-visible spectrum at 550 nm, which corresponds to triangular gold nanoparticles with a size between 20 and 50 nm. This difference may be the reason for the difference in shape and size range of biosynthesized NPs. In addition, the peak around 400 nm is attributed to the Surface Plasmon Resonance (SPR) which is due to collective oscillations of the conduction of electrons of the NPs 25 . TEM analysis. The TEM analysis of the synthesized NPs gives accurate data on the shape, morphology and size of the NPs 26 .The micrographs from the TEM analysis exhibit the spherical shape of the NPs (Fig. 3a,b). In this study, TEM images showed that the NPs are spherical for Au NPs and Ag NPs, respectively. Scientific Reports | (2023) 13:7230 | https://doi.org/10.1038/s41598-023-33632-y www.nature.com/scientificreports/ The calculated average sizes of Au NPs and Ag NPs, detected by the ImageJ (program, ImageJ-win32) were19.61 nm (n = 198) and 24 nm (n = 85), respectively. DLS measurement demonstrated that average size of the NPs were larger than the TEM size, as expected. This size discrimination may reflect the fact that by TEM only the physical size of the nanoparticles is measured, regardless of the coating agent and based on the number of particles, whereas by DLS measurements the hydrodynamic diameter of the nanoparticles is reported to be the same. The diameter of the particle to which the ions or molecules are attached 27 . The ions or other molecules bound to the NPs increase as the size of the NPs increases. As a result, the hydrodynamic diameter of the particles is always larger than the TEM particle size 28 . However, to optimize the size of nanoparticles and their performance in biological experiments, many researchers have shown the importance of the hydrodynamic diameter of the particles 27,29,30 . SEM analysis. In scanning electron microscopy, the morphology of synthesized nanoparticles was approximately spherical shaped (Fig. 4a,b). XRD analysis. The XRD analysis was carried out to verifying the crystalline nature of the as obtained NPs using an X-ray diffraction method Bennur 30 . Figure 5a,b display the XRD pattern of the prepared NPs. Four peaks at 2θ values of 38.2°, 44.5°, 64.8°, and 78° corresponding to (111), (200), (220), and (311) planes of gold and silver were observed. In the present study, the XRD pattern was similar to the one taken in the results of the study done by Khatami 23 .
FTIR spectroscopy analysis. The evaluation of the presence of likely biomolecules on the surface of the as prepared NPs can be evaluated by FTIR analysis 31 . In this work, FTIR spectroscopic studies were carried out to identify possible reducing agents present in essential oil. As shown in Fig. 6a Fig. 6b,c, the peak observed at 2955 is characteristic of the C-H group that exists in monoterpenes structures such as limonene and carvone which can be responsible for reducing and covering gold and silver ions 32,33 . The appearance of the band at 2360 cm −1 can be assigned to the triple bond C≡C presents in the AuNPs. There is a peak at 1676 cm −1 which is indicative of the presence of a stretch C=O bond in some constituents. The lack of a peak at 1144 cm −1 demon-  FTIR spectroscopic studies were also carried out for AgNPs and essential oil to identify possible reducing agents. As shown in Fig. 6a Fig. 6b, the peak observed at 2923 is characteristic of the C-H group that exists in monoterpenes structures such as limonene and carvone. The appearance of the band at 2361 cm −1 can be assigned to the triple bond C≡C presents in the AgNPs. There is a peak at 1676 cm −1 which is indicative of the

DLS analysis.
Twenty-four hours after adding both solutions to the essential oil, DLS analysis was used to analyze the particle size distribution. The AuNPs and AgNPs size range show that they have a non-uniform distribution, with an average particle size of 47.24 nm and 72.38 nm, respectively (Fig. 7a,b).
MTT assay. The cytotoxicity of NPs could be affected by functional groups, surface charge and nanoparticle size. Furthermore, cell type play as a determinant factor in sensitivity to cytotoxic effects 34 . The effect of biosynthesized AuNPs and AgNPs on HEPG-2 cells was assessed by MTT assay, which is shown in Fig. 8. The green synthesized NPs, both represent in-vitro cytotoxic property against HEPG-2 cells. The higher concentrations of both NPs (0.1-2 mg/mL) significantly lower the viability of cells in a dose-dependent manner (Fig. 8). HEPG-2 cell line showed greater sensitivity to AuNPs (IC50 = 0.4834 mg/mL) than AgNPs (IC50 = 0.6145 mg/mL) after exposure for 24 h. (Fig. 9). There are three proposed mechanisms for the anticancer activity of biological NPs. Firstly, the apoptotic pathway, which depends on an increased level of ROS which leads to oxidative stress and DNA fragmentation in the cancerous cell. Secondly, interference of proteins/DNA, resulting in cell chemistry functions. Thirdly, the interaction of biological NPs to cell membranes makes changes in the cell permeability and mitochondrial dysfunction. It has been revealed that the activation of p38 MAPK and Caspase-3 at gene and protein expression levels results in response to nanoparticles 35 . Antibacterial activity. The antimicrobial activity of the prepared gold and silver nanoparticles was evaluated using five different bacteria, including E. coli, L. monocytogenes, S. Typhimurium, S. aureus, and B. cereus. Tables 1 and 2 show that with the increase of the nanoparticles, the diameter of the inhibitory zone increased dramatically. Silver nanoparticles exhibited better antimicrobial activity against the bacteria than AuNPs. Zones AuNPs in the concentrations of 12.5 µg/mL did not show any inhibitory zone around the studied bacteria. The largest inhibition was observed by silver nanoparticles against E. coli.
The antimicrobial effect of Silver has long been known in medical and industrial processes. One of The most important applications of silver and silver nanoparticles in medicine is in topical ointments to prevent infection of burns and open wounds 38 . The other outcomes of silver nanoparticles is cancer treatment that is in relationship with their fabulous properties such as alteration in metabolic activity, inducing of reactive oxygen species (ROS) and gene alteration in cancer cells 39 .
The antibacterial action of silver nanoparticles can be related to the reaction of metal ions with thiol and sulfhydryl groups in the bacterial cell membrane protein. This reaction can reduce the permeability of the bacterial cell leading to destruction of cellular respiration and, consequently, cell death. Besides, Ag can bind to bacterial DNA, causing denaturation and inhibiting replication 4 . Due to containing great number of silver atoms in silver nanoparticles they can act as an antibacterial substance in both gram positive and gram negative bacterias by penetrating into the pathogen's cells and led to DNA gyrase blocking 40 .
In accordance with the findings of niloufar work, antibacterial activity of AgNPs can be attributed to the ROS (reactive oxygen species) of bacterial cell. Accordingly AgNPs could harm DNA and membrane proteins by enforcing the release of ROS. Generally, antibacterial activity of silver nanoparticles have been reported previously and demonstrated that silver nanostructures can cause cell damage by accumulating in cell cytoplasm, following the coagulation with sulfur and phosphorus containing compounds in order to deactivate the intracellular enzymes. These functionalities of silver nanoparticles can be contributed in bacterial cell lysis by coagulation, due to owning of sulfur and phosphorus in bacteria's cell membrane, DNA and proteins of the membrane 41 .
Also, AuNPs have antimicrobial activity through binding to the surface of microorganisms, causing damage to the flagella and destruction of the cell walls 42 .
Gold nanoparticles exhibited anticancer activity through colorectal cancer cells by various mechanisms that led to cell death. In addition, Barabadi revealed that AuNPs express potential anticancer activity against cervical cancer cells 43 .
As reported in Barabadi et al. 22 , study, the AgNPs that synthesized via green method demonstrated higher antibacterial activity in compromise with chemically synthesized AgNPs. The antibacterial activity of metallic NPs can be explained by several mechanisms. One of the main mechanisms for their bactericidal action is the oxidative dissolution of these particles. In an oxygenated medium, nanomaterials act as reservoirs of metallic ions which turn into a potential antibacterial agent. Furthermore, the components of essential oil affect the antibacterial activity of the synthesized nanoparticles by damaging the bacterial cell membrane 36 .
The results of this study provide an important basis for the application of silver and gold nanoparticles produced using the essential oil of M. spicata in the treatment of human infections associated with the microorganisms used in this study.   www.nature.com/scientificreports/ Figure 10 shows that the DPPH radical scavenging activity was increased with increasing of NPs concentration. This result is in good agreement with the previous reports in the literature 21,[44][45][46][47] . The highest inhibition of 38.90% and 57.20% was observed at 100 μg/mL by AuNPs and AgNPs, respectively.
The antioxidant capacity of AuNPs and AgNPs was further evaluated by ABTS scavenging. In the ABTS assay, the AuNPs and AgNPsalso showed a dose-dependent activity (Fig. 11). Synthesized nanoparticles exhibited higher antioxidant activity than MSEO in both assays. These results suggest the good antioxidant activity of the synthesized nanoparticles. Also, AgNPs had superior activity to AuNPs in both tests. The difference in the attached functional groups of MSEO to nanoparticles may be responsible for the differences in their scavenging activity. These results agreed with the previous result reported by Adebayo 44 where the antioxidant activity of the synthesized gold and silver nanoparticles from Opuntia ficus-indica extract was evaluated using Nitric Oxide (NO). Higher DPPH radical scavenging activity was also reported By Huo 45 in silver chloride nanoparticles of Glycyrrhiza uralensis (Gu-AgClNPs) when compared to gold nanoparticles of Glycyrrhiza uralensis (Gu-AuNPs). Surface morphology could affect the biological activity of nanoparticles. In a study spherical shaped of synthesized silver nanoparticles represented high antioxidant activity and that was in agreement with our findings 41 . The antioxidant activity of the prepared nanoparticles has been observed to be a result of the absorption or integration of more bioactive compounds or bioreductant molecules of the plant on the surface of the nanoparticles which Table 1. Diameter of inhibition zone (mm) produced by silver nanoparticles (AgNPs) against various bacteria. Values are Mean ± SD (n = 3). The different letters (a-b) indicate a significant difference (P < 0.05) among various bacteria.  www.nature.com/scientificreports/ increases the surface area for radical scavenging activity (Fig. 10). Hence, the reducing effect of the nanoparticles can be attributed to the phenolic functional groups on the surface of nanoparticles 44 .

Conclusion
In this study, a low-cost and simple method was used to synthesize AgNPs and AuNPs using essential oil from Mentha spicata through the reduction of Ag + and Au 3+ ions. This might be due to the presence of phenolic and flavonoid compounds in essential oil as electron donors. The formation of AgNPs and AuNPs was confirmed using UV-Vis Spectroscopy, dynamic light scattering, SEM, TEM, FT-IR, and XRD.The micrographs from SEM and TEM analysis proved the formation of small size and spherical shape of the nanoparticles. FTIR analysis showed that biologically active compounds such as monoterpenes could assist in the formation and stabilization of both types of nanoparticles. XRD analysis showed nano-metal structures in particles. The synthesized nanoparticle exhibited a strong cytotoxic, antibacterial and antioxidant activity. The AgNPs showed higher antioxidant and antimicrobial activities than AuNPs. However, the cytotoxicity study showed that the HEPG-2  www.nature.com/scientificreports/ cell line was more sensitive to AuNPs than AgNPs after 24 h of exposure. The procedure for the biosynthesis of nanoparticles has several advantages such as cost-effectiveness, compatibility for biomedical and pharmaceutical, and food applications as well as for large-scale commercial production. In the future, it would be significant to select such plants' EO compounds, to understand the clear mechanism of biosynthesis, and to technologically improve the nanoparticles to achieve better control oversize, shape, and absolute mono dispersivity to utilize the potential of herbal medicine in nanoscience for biomedical applications.

Methods
Essential oil. The commercial Mentha spicata EO used in this study was purchased from Barij Essence Co.
Ltd. (Kashan, Iran). The plant essential oil was kept at a temperature of 4-6 °C before analysis. The essential oil was used as a reducing agent as well as a stabilizing agent for producing NPs.
Gas chromatography: mass spectrometry. The gas chromatography-mass spectrometry (GC-MS) was accomplished using an Agilent 7890/5975C GC-MS system, fitted with a DB-624 capillary column (30 m length × 0.25 mm; 0.25 µm film thickness) for analysis of the essential oil. The column temperature program was adjusted as follows: the initial temperature of the oven was set to 60 °C and held at this temperature for 3 min. The temperature was then increased to 220 °C at a rate of 5 °C per min. and held for 1 min. The temperature of the injector was 250 °C. The amount of injection was 0.2 μl. The carrier gas was Helium with a flow rate of 1.2 mL/min and a split ratio equal to 1:4. The mass spectrometer was operated in EI ionization mode at 70 eV, and complete scans from 40 to 350amu (atomic mass units) were recorded. The constituents of the essential oil were identified by the confirmation of the experimental gas chromatographic retention indices (RI) relative to n-alkanes (C8-C24) and mass fragmentation with those of the National Institute of Standards and Technology (NIST 08) commercial library, as well as with literature data 46 . All analyses were carried out in triplicate.
Green synthesis of nanoparticles. Chloroauric acid (HAuCl 4 ) and silver nitrate (AgNO 3 ) purchased from Sigma-Aldrich, Germany was used in the test. Thus, 0.5 mL of the essential oil was mixed with 19 mL of HAuCl 4 and AgNO3 solutions separately (10 mmol/mL) in a conical flask and then a magnetic stirrer is kept inside the conical flask and started to run vigorously on the hot plate. They were incubated at room temperature for 24 h. The reduction of gold ions was initially confirmed by visual inspection of color change from pale yellow to ruby-red (AuNPs) and light brown (AgNPs) Then by UV-visible spectrophotometer at different times (0, 30 min, 2 h, 4 h and 24 h). To separate the unreacted essential oil, the sample was centrifuged at 6000 rpm for 11 min. The sediment left at the end of the tube was resuspended in distilled water. The water was evaporated in a furnace. The thin layer of NPs remaining on the watch glass surface was used for further studies 18 . Cell lines and culture conditions. The HEPG-2 cell line was acquired from the National Cell Bank of Iran (Pasteur Institute, Tehran, Iran) and cultured in 75 cm 2 culture flasks using DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco Inc., USA) and streptomycin-penicillin (100 µg/mL and 100 IU/ mL, respectively) in an incubator providing 5% CO 2 and 95% humidity at 37 °C. Every 2 days culture medium was replaced with fresh medium until reaching suitable confluency of about 70%. Then they were detached with Gibco Trypsin-EDTA (0.25%) and subcultivated.

Characterization of
MTT assay. The density of viable cells was determined by Trypan blue exclusion assay by using a hemocytometer and the preparation was diluted with the DMEM medium to yield optimal plating densities for cells 47 before the MTT assay.HEPG-2 cells were seeded in a 96-well flat bottom microtiter plate at a density of 12 × 10 3 cells/well and allowed to adhere. After 48 h of incubation and producing the confluent cells, the stock solution of AuNPs and AgNPs was freshly prepared and diluted with the cell culture medium to the desired concentrations (0.1-2 mg/mL). After 24 h of exposure, the cells without treatment were used as control and received only the culture medium as the final concentration. The cytotoxicity test was carried out by MTT assay according to the manual of the manufacturer for investigating changes in mitochondrial/non-mitochondrial dehydrogenase activity. 50 µl of 2 mg/mL MTT solution (Sigma Aldrich, Germany) was added to each well and the plate was incubated for 4 h at 37 °C in a CO 2 incubator. The medium was then aspirated, and the formed formazan crystals were solubilized by adding 100 μl of DMSO per well for 10 min at 37 °C in a CO 2 incubator. Following half an hour, optical density (OD) was defined at 570 nm in a microplate reader (Tecan, Switzerland). Cell viability was expressed based on the absorbance of the control wells, which were considered as 100% of absorbance. Cytotoxicity was expressed as the concentration of the substance (both AuNPs and AgNPs) inhibiting cell growth . The bacteria were transferred from a sterile microtube to brain heart infusion (BHI) agar (Merck, Germany) and incubated at 37 °C for 24 h. The second culture was prepared from the first culture and incubated under the same condition. Serial dilutions were prepared from the bacterial culture using the tubes containing 9 mL of sterile peptone water (0.1%, v/w). Then, 0.1 mL of each dilution was cultured on the surface of Muller Hinton agar (Merck, Germany) to determine the bacterial concentration. The antimicrobial effect of nanoparticles was studied against the selected bacteria by well diffusion technique. First, the bacterial suspension (with a final concentration of 10 5 CFU/mL) was cultured on the surface of Muller Hinton agar. Then, wells with a diameter of 6 mm were created on the culture medium. 50 µL of synthesized nanoparticles in different concentrations (100, 50, 25, 12.5 µg/mL) were transferred into the wells under sterile conditions. After incubating the plates at 37 °C for 24 h, the diameter of the inhibition zone around the wells was measured.
Antioxidant effect. The antioxidant effect of nanoparticles to inhibit was evaluated using DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical as described by Maciel 48 . For this purpose, an aliquot of 1.5 mL different concentrations of nanoparticles (100, 50, 25, 12.5 and 6.25 µg/mL) was mixed with 1.5 mL of DPPH solution (0.2 mmol L −1 ). Then, the above mixture was completely homogenized and incubated at 24 °C in the dark for 30 min. The absorbance of the mixture was measured at 520 nm. Butylated hydroxytoluene (BHT) and ascorbic acid will be used as a standard.
To evaluate the scavenging activity of nanoparticles, the ABTS •+ assay was also used according to the method described by Bakur 49 . The free radicals of ABTS •+ were prepared by mixing the stock solution of ABTS (2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) (0.7 μM) and potassium persulfate (2.45 μM). The mixture was incubated in a dark environment at room temperature for 16 h. The solution containing ABTS •+ free radicals was diluted with 80% ethanol until its absorbance reached 0.700 ± (0.05) at 743 nm. Then, 200 µL of ABTS •+ solution was mixed with 20 µL of nanoparticles at different concentrations (100, 50, 25, 12.5, and 6.25 µg/mL). After incubation for 6 min in a dark place, the absorbance of the samples was measured at 734 nm using ascorbic acid and BHT as standard. Antioxidant activity of DPPH and ABTS •+ radicals by AgNPs and AuNPs were calculated according to the following formula: where Abs sample is the absorbance of sample/standard mixed with DPPH radical in methanol; Abs blank is the absorbance of methanol and Abs control is the absorbance of DPPH radical in methanol; Abs blank is the absorbance of methanol.
Statistical analysis. Data were expressed as mean ± standard deviation (SD) of three independent tests.
The statistical difference in data was evaluated using independent t-test and one-way ANOVA test using SPSS software version 19.0. P values < 0.05 was considered statistically significant.

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Data availability
All data used to support the conclusions of this study are included within the article.